This invention relates to the integrated, multi-dimensional, rapid analysis of solutions of a large number of mixed molecular species, commonly called xe2x80x9clibrariesxe2x80x9d. More specifically, the invention is directed toward methods for the discovery of molecular entities useful in a variety of biological contexts using hi-flux screening of natural and synthetic libraries to select ligands having a desired affinity for a target molecule of interest.
Multi-dimensional systems, i.e., systems involving the application of multiple distinct physico-chemical separation steps, are known to be useful for many applications. The purification of proteins, for example, frequently is accomplished using multiple passes through different chromatographic columns exploiting differential partitioning such as adsorption and size exclusion. Inherent in any multi-dimensional process is the necessity to identify the desired component, from the output of the first partition, to collect it, and to introduce it into the next dimension of the system. Disadvantages of such systems include slow analysis, solvent incompatibility between successive partitioning phases, the necessity of labor intensive handling, and consequent contamination or loss of sample.
The recent prior art discloses various new methods for implementing the search for novel agents such as, for example, pharmacological or therapeutic agents (i.e., drug discovery) agents useful in animal care or management, agriculturally useful chemicals, selective biocides for insects, weeds, or other pests, and catalytic and other entities useful in industrial processes. Collections of molecules or xe2x80x9clibrariesxe2x80x9d are prepared and screened for molecules having a specified bioactivity, as indicated initially by detection of binding between one or more species or xe2x80x9cligandsxe2x80x9d in the library and a xe2x80x9ctargetxe2x80x9d molecule with which it reacts to influence some biological process. More specifically, libraries consist of a complex assortment of molecules containing one or more ligands which may bind to a target of interest. The identification of ligands which bind may provide a lead for identifying compounds with a desired biological activity, e.g., as a potential drug candidate. As methods have become available to screen these complex mixtures more effectively, interest in exploiting this new xe2x80x9crational designxe2x80x9d or xe2x80x9cdirected molecular evolutionxe2x80x9d approach has increased.
Libraries of biopolymers may be prepared by the sequential synthesis based on randomized addition of amino acid, nucleotide, or sugar residues, or combinations thereof, to form peptides, RNAs, polysaccharides, glycosaminoglycans or the like, thereby to prepare a random mixture of oligomers. Techniques suitable for preparing protein or peptide libraries at the nucleic acid level by phage display and similar technologies also are known. Likewise, these general synthesis approaches could be adapted to prepare peptide nucleic acid (PNA) libraries, or libraries of PNA/DNA or PNA/RNA chimeras, and indeed other complex mixtures of synthetic molecules.
Screening of soluble peptide libraries frequently is performed either by immunoassay or by laboriously assaying for a particular biological function (e.g. blocking of viral replication). These methods are not necessarily target based and in most cases involve tedious set up. See Scott and Craig, Curr. Opin. Biotech. 5, 40-48 (1994); Dooley et. al. Proc. Natl. Acad. Sci., 90:10811-10815 (1993); Dooley et. al., Life Sciences, 92:1509-1517 (1990); Houghton et. al., Biorg. Med. Chem. Lett., 3:405-412 (1993). For example, inhibitors of HIV protease have been identified by screening sets of equimolar peptide mixtures, together containing more than 240,000 soluble tetrapeptides. See Owens et. al. Biochem. Biophys. Res. Comm., 181:402-408 (1991). It has also been suggested to use a phosphopeptide library to determine the sequence specificity of the peptide-binding sites of SH2 domains by employing the GST-SH2 fusion protein immobilized onto a column. See Songyang et al. Cell, 72, 767-778 (1993).
The screening methods described immediately above are based upon identifying which ligand in a mixture binds to a target of interest. Binding typically is assayed with either the ligands of the library or the target immobilized on some form of solid support. Various solution parameters may be adjusted to emulate different binding conditions and to obtain different ligands. Often, peptides which are obtained through procedures involving their immobilization to a support have disappointing affinity, i.e., have a binding constant too low to be useful. Traditionally, antibodies are used for the affinity purification of proteins and other biomolecules. However, the cost of generating antibodies, the potential for antibody leaching, and the need for relatively harsh eluting conditions pose problems for the routine use of antibodies in affinity purification.
Screening methods known in the art thus are not entirely satisfactory. Prior methods for detecting or identifying ligands which bind to a target of interest often fail to provide ligands of sufficiently high affinity to be useful, and additionally suffer from the loss of sample, the need for large amounts of ligands, and the need to vary loading, binding, or elution conditions to obtain useful results. Additionally, existing systems are unable selectively to screen a library while simultaneously determining the affinity of selected ligand(s) for the target under relevant conditions.
A major hurdle in the exploitation of current screening techniques of the type described above is effective chemical characterization of ligands identified in these processes. Chemical characterization, e.g., determining the sequence of an identified biopolymer, is at best time-consuming and complex. A major focus of prior art screening techniques is to enable the collection of enough of or enough information about a ligand of interest so as to permit determination of its structure and to enable synthesis of larger amounts for testing and further empirical structural refinement.
Accordingly, there is a need for integrated, multi-dimensional screening, selection and analysis systems and methods which permit automated, direct transfer of samples without dilution or loss between various dimensions, and efficiently screen for, and subsequently permit characterization and recovery of ligands to a target of interest, even when present at low concentration.
Accordingly, the present invention is directed to rapid, efficient and automated, multi-dimensional systems, methods and apparatus for screening libraries to select, recover and characterize a candidate ligand with a desired or preselected affinity K for a preselected target molecule. Additionally, the present invention is directed to certain combination of individual dimensions of such a system, which can be used to obtain a desired result, and, specifically to a method of detecting a ligand to a target of interest which overcomes the disadvantages of the methods known in the art.
Additional features and advantages of the invention will be set forth in the description which follows, and in part will be apparent from the description and drawing, or may be learned by practice of the invention. The objectives and other advantages of the invention will be realized and attained by the process particularly pointed out in the written description, drawing, and appended claims.
To achieve these and other advantages, and in accordance with the invention as embodied and broadly described, the invention provides novel methods for screening a sample to select a ligand to a target of interest and for obtaining information about the ligand and its binding characteristics. Specifically, the claimed multi-dimensional methods involve combining a solution of heterogeneous ligands with the target of interest to screen the ligands on the basis of one or more binding characteristics. Ligands having the first binding characteristic will bind to the target of interest to form a target/ligand complex. The complex then optionally is separated from the unbound components using any of a variety of separation techniques, e.g., size exclusion. At least one of the complex or unbound components then is introduced to a second xe2x80x9cdimensionxe2x80x9d. The second dimension is capable of separating components based upon a second binding characteristic. One then elutes the ligand having the desired binding characteristics.
Additionally, the invention relates to a method of detecting the presence of a ligand having a desired or preselected affinity (K) for a preselected target molecule in a sample of ligands in a solvent by loading a column with a known concentration of target molecules (T), and passing the sample through the column so that ligands in the sample bind to the column through the target molecule. A series (n) of column volumes of solvent then are passed through the column, where n is a number of column volumes between 1 and 10,000. A subset of the column volumes exiting the column is passed through a ligand accumulator to immobilize on the accumulator ligands having the preselected affinity K. Ligands having the preselected affinity then can be eluted from the accumulator, and, optionally, identified, and/or synthesized in commercial quantities.
In another aspect, the invention relates to a method of separating mixed species of ligand dissolved in a solvent into separate fractions of ligands, wherein each fraction is characterized by a different affinity or range of affinities for a preselected target molecule. Initially, the mixed ligand species are passed through a column comprising immobilized target molecules so that the ligands will bind to the target. A series of column volumes of solvent then are passed through the column, and at least two subsets of the column volumes of solvent exiting the column are then passed through a ligand accumulator, thereby immobilizing ligands characterized by separate ranges of affinity constants. The fractions containing ligands characterized by different ranges of affinities are then optionally eluted from the accumulator to separate them chemically for further screening or analysis.
In yet another embodiment, the invention relates to a multi-dimensional system or apparatus for obtaining and identifying ligands having a preselected affinity for a target of interest. The multi-dimensional system consists of at least two dimensions, the first comprises a chromatographic element to which a concentration, preferably a known concentration T, of target molecules of interest is bound. The system has as a second dimension another chromatographic element followed by a detector. Additionally the system in some embodiments has an interface between each dimension, and a controller for automatically regulating the various dimensions of the system.
In yet other embodiments, the apparatus comprises multiple valves, a first column with target molecules immobilized thereon; an accumulator or separate column to receive at least a portion of the exist stream of the first column, an optional interface to condition the exit stream to make it compatible with the accumulator or second column, and a detector such as a mass spectrometer. The interface may, in some embodiments include a buffer exchange such as a mixed bed ion exchanger, a cation exchanger or an anion exchanger, and means to inject solvent so that the pH, ionic strength, etc. can be controlled so as to permit further downstream partitioning of partly screened ligand species.
The ability of the methods and apparatus of the invention to provide for continuous flow through multiple partitioning dimensions is dependent in many cases on the use of interface columns. These condition the solvent containing the dissolved ligands exiting an upstream column for effective partitioning in a downstream column. In one such interface, effluent high in salt is desalted by passage through a reverse phase column. The ligands adsorb, the salt is washed out, and the ligands then are eluted with salt-free or low salt solvent. In another, organic solvent such as acetonitrile is removed by passing the solution through an ion exchange column, binding the ligands therein, and subsequently eluting with an aqueous eluant. In still another, the pH of acidic solvents is increased by binding in a cation exchange resin, washing out the acid, and eluting in, e.g., a neutral pH solvent. Similarly, the pH of alkaline solvents may be decreased by binding in an anion exchange resin.
In yet other embodiments, the invention features an interface for sampling a liquid chromatographic (LC) exit stream, and delivering the sample to a mass spectrometer (MS). The sampler has a predetermined sample volume disposed, for example, in a sample loop, alternatively switchable to extract from an LC exit stream, and to insert into an analysis stream of an MS. A sample controller cycles the sampler to first extract and then to insert the sample. In various embodiments, the sampler can comprise a multi-port valve and the sample volume is disposed within tubing of a predetermined volume. The sample controller may cycle the sampler to take a sample of the LC eluate a plurality of times during an LC analysis peak. Other embodiments may include a second sampler. The first and second samplers can be placed in series.
The embodiments of the methods, apparatus and system of the invention described above may optionally include a detector for identifying a selected ligand. The detector may consist of, for example, a mass spectrometer or a fluorescence detector.
Additionally, in other embodiments, the invention relates to a method of detecting a ligand having a desired high affinity K for a preselected target molecule when the ligand and the target are present together in preselected solvent conditions. The ligand to be detected may be one of a multiplicity of ligand species in a heterogeneous sample. The method involves immobilizing the target molecule onto a column, passing the sample through the column to promote binding of ligands in the sample to the target molecules, and then passing a series of column volumes of a solvent defining the solvent conditions. A subset (kp) of the column volumes exiting the column are then passed through a ligand accumulator to immobilize thereon ligands having the desired affinity, and then those ligands are eluted. A selected ligand may be characterized by a high affinity K for the target molecule equal to approximately (kp)/T, where T is the concentration of target molecules in the column. In some instances, the conditions under which the sample is passed through the column (i.e. to promote binding of ligands to target molecules) is different from the preselected solvent conditions.
In other embodiments, the methods of the invention relate to the detection of a ligand having a high on-rate, Ko, when said ligand and said target molecule are present together in preselected solvent conditions. The ligand is detected in a sample comprising multiple ligand species, at least one of which binds a preselected target molecule with an affinity of at least about 104Mxe2x88x921. The target molecule is immobilized onto a column, and the sample is provided in a sample solution defining the preselected solvent conditions. The sample is passed through the column at a high linear fluid velocity so as to minimize the residence time of the ligands in the column, thus selectively binding high on-rate ligands to the target molecules, in preference to other ligands in the sample. One can then elute the column to obtain an output, and identify the high on-rate ligands. Optionally, the output can be passed through a ligand accumulator, which is then eluted to produce an output rich in a high on-rate ligand.
In other embodiments, the invention relates to a method of selecting ligands to a target of interest on the basis of the off-rate of the ligand.
The methods of the invention in certain aspects relate also to a method for detecting a ligand having a high affinity for a target molecule by providing a library obtained by the digestion of one or more proteins or other biopolymers. The sample solution and a target molecule are combined under conditions which allow suitable ligands, if present, to bind to the target; and thereafter the ligands which bind to the target (forming a complex) are separated from those which do not bind. The sample solutions may be obtained by the digestion of any protein, including post-translationally modified proteins, antibodies, etc.
The methods of the claimed invention also relate to detecting a ligand in a library which will bind when the ligand and the target molecule are present together in preselected solvent conditions, e.g., physiological saline. As before, a target molecule is immobilized onto a column, and the sample is passed through the column under the preselected solvent conditions. Next, a series of column volumes of solvent is passed through the column to select a desired ligand. The eluate is then introduced to a ligand accumulator.
The methods also relate to the preparation of pharmaceutically active compositions using the multi-dimensional methods described above, and to the subsequent commercial production of such compositions.
In yet other aspects, the invention relates to methods of selecting ligands based upon one or more binding characteristics by the use of multiple dimensions.
In an important aspect, the invention provides apparatus and methods which are automated, fast, and operate by continuous flow. The methods are capable in preferred embodiments of selecting ligands having affinity and specificity for essentially any target molecule, separating the members of the select group from one another, and obtaining physico-chemical data characteristic of the structure of the selected ligands. The nature of the library useful in the system essentially is unlimited. Thus, mixtures of organic compounds may be used. Digests of biopolymers, either natural or synthetic, are particularly attractive. Such digests may comprise mixtures of peptides, polysaccharides, polynucleotides, various derivatized forms thereof, and variously sized fragments thereof. The biopolymers may be extracted from plant or animal tissues, diseased or healthy, digested if necessary, or used as is. Such libraries are available in abundance, easy to prepare, may be of lower toxicity and more stable than synthetic peptides, and may be varied and screened systematically.
It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory and are intended to provide further explanation of the invention as claimed.